Fluorescence lifetime study of ECFP/EYFP labeled MBP in Förster resonance energy transfer

This paper studies the conformational change of the binding protein by a fluorescence lifetime method. As a model protein, maltose binding protein (MBP) where enhanced cyan protein (ECFP) and enhanced yellow fluorescent protein (EYFP) were genetically fused to act as a donor and an acceptor in Förster resonance energy transfer (FRET) was used. The ECFP and the EYFP were linked to the C-terminal and N-terminal regions of the MBP, respectively. In order to investigate the conformational change of the MBP, the lifetime of the ECFP, which acts as a donor in the ECFP:MBP:EYFP fusion protein, was analyzed during the FRET process. We observed that two lifetime components exist when the ECFP is linked to the MBP and that the lifetime of the ECFP is shortened when ECFP:MBP:EYFP protein undergoes a conformational change as a result of the maltose binding. In addition, we observed that the lifetime of the donor is gradually shorter in the ECFP:MBP:EYFP fusion protein as the maltose concentration increases. By a lifetime analysis and simulation study, we found that the participant rate of the ECFP:MBP:EYFP protein in FRET is the main cause of the donor lifetime shortening in relation to the increase of the maltose concentration.     

Study of Macromolecular Chain Dynamics in Polymer Complexes by Time-Resolved Fluorescence Spectroscopy

Poly(methyl methacrylate)s labeled with the anthracene fluorophore were prepared by free radical, anionic, and coordination polymerization yielding atactic and syndiotactic polymers. Unlabeled isotactic poly(methyl methacrylate) was prepared by anionic polymerization. Time-resolved fluorescence spectroscopy was used to study polymer association in solution. The time-dependent decays of fluorescence anisotropy show that stereocomplexation causes an increase in rotational correlation times of anthracene fluorophores both embedded in the polymer backbone and attached at the end of the side chain of polymer molecules. The rotational correlation time of anthracene fluorophore in dimethylformamide as a part of stereocomplex is 11.9 and 30 ns in the side chain and embedded in the polymer backbone, respectively, and shorter than 3 ns in noncomplexing solvent.     

Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with stick boundary conditions.     

Time-resolved fluorescence anisotropy imaging applied to live cells

We have developed a wide-field time-resolved imaging system to image quantitatively both the fluorescence lifetime and the rotational correlation time of a fluorophore. Using a polarization-resolved imager, we simultaneously image orthogonal polarization components of the fluorescence emission onto a time-gated intensified CCD. We demonstrate imaging of solvent viscosity variations through the rotational correlation time of fluorescein in a multiwell plate and apply this technique to probe the microviscosity in live cells.     

Luminescence and anisotropy decays ofN-3-pyrene maleimide labeling IgG proteins and cells

The Py.M (N-3-Pyrene Maleimide) is a dye that covalently binds to reactive amino or sulfhycryl groups to give highly fluorescent protein conjugates. Measurements of luminescence lifetimes and anisotropy decays have been performed with a Phase and Modulation Fluorometer. Complexes of Py.M-antibody (IgG antimouse) and tumoral cells C6 labeled with Py.M have been investigated. The Py.M fluorescence in buffer solution and the protein and cells natural fluorescence have been checked. For Py.M-IgG and labeled cells, the fluorescence decays present interesting behaviours. The least-squares analysis of the experimental results on Py.M-IgG complex points out two lorentzian distributions centered at 74 ns and 11 ns, on the contrary, for the labeled cells, a discrete component at 100 ns and a lorentzian distribution centered at 5 ns are shown. In both systems a weak component lower than 1 ns is observed. The fluorescence decays, mainly the long lifetime one, are very sensitive to oxygen quenching, showing the high efficiency of O₂ quenching. For samples N₂ bubbled, the lifetime experimental resuits show a decrease of the oxygen accessibility from free probe in solution to Py.M-IgG complex and to labeled cells, compatible with a more compact packing of the probe binding site. The experimental results of anisotropy decays of degassed samples show for Py.M-IgG complexes a long rotation correlation time of about 200 ns at T=5°C, assigned to overall rotation of the protein, besides shorter correlation times attributable to inner protein motions. For labeled cells, the long rotation correlation time becomes of the order of 580 ns confirming a progressive increase of the stabilization of the binding site.     

Dynamics of tRNAtyr Probed with Long-Lifetime Metal-Ligand Complexes

The metal-ligand complexes, [Ru(bpy)₂(dppz)]²⁺ (bpy = 2,2??-bipyridine, dppz = dipyrido[3,2-a:2??,3??-c]phenazine) (RuBD) and [Ru(phen)₂(dppz)]²⁺ (phen = 1,10-phenanthroline) (RuPD), display favorable photophysical properties including long lifetime, polarized emission, and very little background fluorescence. To check if RuBD and RuPD reflect the overall rotational mobility of small nucleic acid, we measured the intensity and anisotropy decays of RuBD and RuPD when intercalated into tRNA^tyr using pBC SK(+) phagemid as a control. We used frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. We observed shorter lifetimes for tRNA^tyr than those for the pBC SK(+) phagemid for both probes, however, RuPD showed much larger decrease in the mean lifetime values (64%). The slow rotational correlation time of RuBD (31.3 ns) and the fast rotational correlation time of RuPD (26.0 ns) reflected the overall rotational mobility of tRNA^tyr. In addition, the steady-state anisotropy and time-resolved anisotropy decay data showed a clear difference between tRNA^tyr and pBC SK(+) phagemid. This suggests the possibility of a homogeneous assay for identifying target nucleic acids and/or nucleic acid binding proteins.     

Translational and Rotational Motions of Albumin Sensed by a Non-Covalent Associated Porphyrin Under Physiological and Acidic Conditions: A Fluorescence Correlation Spectroscopy and Time Resolved Anisotropy Study

The interaction between a free-base, anionic water-soluble porphyrin, TSPP, and the drug carrier protein, bovine serum albumin (BSA) has been studied by time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS) at two different pH-values. Both rotational correlation times and translational diffusion times of the fluorescent species indicate that TSPP binding to albumin induces very little conformational changes in the protein under physiological conditions. By contrast, at low pH, a bi-exponential decay is obtained where a short rotational correlation time (phi (int) = 1.2 ns) is obtained, which is likely associated to wobbling movement of the porphyrin in the protein binding site. These physical changes are corroborated by circular dichroism (CD) data which show a 37% loss in the protein helicity upon acidification of the medium. In the presence of excess porphyrin formation of porphyrin J-aggregates is induced, which can be detected by time-resolved fluorescence with short characteristic times. This is also reflected in FCS data by an increase in molecular brightness together with a decrease in the number of fluorescent molecules passing through the detection volume of the sample.     

Acridones and Quinacridones: Novel Fluorophores for Fluorescence Lifetime Studies

Two new families of fluorescent probe, acridones and quinacridones, whose fluorescence lifetime can be altered to produce a range of lifetimes from 3 ns to 25 ns are described. Both families of fluorophore have fluorescence lifetimes which are unaffected by pH in the range of 5 to 9 and show a marked resistance to photobleaching. The probes have been modified to allow them to be attached to biomolecules and the labelling of a neuropeptide (substance P) is described. The labelled peptides have the same fluorescence lifetime as the free fluorophore. Quinacridone, with an emission around 550 nm offers a long fluorescence lifetime, photostable alternative to fluorescein.     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

Time variation of fluorescence lifetime in enhanced cyan fluorescence protein

The lifetime variations of enhanced cyan fluorescence protein (ECFP) in relatively short integration time bins were studied via time-correlated single photon counting (TCSPC) measurement. We observed that minimum photon counts are necessary for the lifetime estimation to achieve a certain range of variance. The conditions to decrease the variance of lifetime were investigated and the channel width of the measurement of TCSPC data was found to be another important factor for the variance of lifetime. Though the lifetime of ECFP is best fit by a double exponential, a mono exponential fit for the same integration time is more stable. The results may be useful in the analysis of photophysical dynamics for ensemble molecules in short measurement time windows.     

Fluorescence anisotropy decay study of self-association of bacterial luciferase intermediates

The fluorescence dynamics parameters of the fluorescent transient flavin-luciferase species from the typesVibrio fischeri andPhotobacterium leiognathi are presented. The fluorescence anisotropy decay is a single exponential function for both types. The correlation time is 70 ns for theP. leiognathi fluorescent transient intermediate (2°C, aqueous buffer, pH 7.0), consistent with the rotational correlation time of the luciferase macromolecule (77 kD) to which the flavin fluorophore is rigidly attached. In contrast, for theV. fischeri species the observed correlation time for the anisotropy decay function is 133 ns. This suggests that protein self-association occurs in theV. fischeri case and this is confirmed by filtration, where the fluorescent transient fromV. fischeri does not pass through a 100,000 molecular weight cutoff membrane, whereas theP. leiognathi species does. The filtration method also demonstrates self-association in the luciferase peroxyflavin and photoflavin fromV. fischeri. A monomer-dimer equilibrium also explains the previously reported high correlation times for theV. harveyi luciferase-flavin species. It is proposed that the self-association competes with the lumazine protein interaction in the bioluminescence reaction.     

New Insights in the Interpretation of Tryptophan Fluorescence

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore–environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.     

Probing the Sol-Gel Transition in SiO2 Hydrogels—A New Application of Near-Infrared Fluorescence

Picosecond time-resolved fluorescence spectroscopy has enabled us to use a near-infrared fluorescent dye to probe the sol-gel transition in SiO₂ hydrogels, polymerized from sulfuric acid and sodium silicate solution, for the first time. We compare the microviscosity surrounding the probe during the sol-to-gel transition as predicted by two alternative models which both describe the decay of fluorescence anisotropy well. The results for one rotational time and a residual anisotropy imply that macrogelation of the sol leads to relatively small changes in the mobility of the fluorophore caused by small changes in microviscosity, but after much longer times, e.g., 1500 min, the mobility of the fluorophore decreases, reflecting a rapid increase in microviscosity of over several orders in magnitude. In sharp contrast, analysis of the anisotropy in terms of two rotational times predicts little change in microviscosity over the whole polymerization process.     

Dynamics of Bacteriophage R17 Probed with a Long-Lifetime Ru(II) Metal-Ligand Complex

The metal-ligand complex, [Ru(2,2′-bipyridine)₂(4,4′-dicarboxy-2,2′-bipyridine)]²⁺ (RuBDc), was used as a spectroscopic probe for studying macromolecular dynamics. RuBDc is a very photostable probe that possesses favorable photophysical properties including long lifetime, high quantum yield, large Stokes’ shift, and highly polarized emission. To further show the usefulness of this luminophore for probing macromolecular dynamics, we examined the intensity and anisotropy decays of RuBDc when conjugated to R17 bacteriophage using frequency-domain fluorometry with a blue light-emitting diode (LED) as the modulated light source. The intensity decays were best fit by a sum of two exponentials, and we obtained a longer mean lifetime at 4 °C (<τ> = 491.8 ns) as compared to that at 25 °C (<τ> = 435.1 ns). The anisotropy decay data showed a single rotational correlation time, which is typical for a spherical molecule, and the results showed a longer rotational correlation time at 4 °C (2,574.9 ns) than at 25 °C (2,070.1 ns). The use of RuBDc enabled us to measure the rotational correlation time up to several microseconds. These results indicate that RuBDc has significant potential for studying hydrodynamics of biological macromolecules.     

Radiationless deactivation of hybridization-sensitive DNA probe

In this study we attempt to determine the deactivation mechanism of a hybridization-sensitive DNA probe by using steady state absorption and fluorescence spectroscopy. Thiazole orange or 2,2′-Methylenebis(N-alkylbenzothiazole) is covalently linked to a single nucleotide in the DNA probe. Radiationless deactivation of the fluorophores is mainly determined by photo-induced isomerization of the methine bridge. Experiments done using solutes of differing molecular weight (sucrose, glucose and glycerin) to provide solutions of similar viscosity reveals the importance of solution viscosity and fluorophore conformation on radiationless deactivation. The quantum yield of the fluorophore in high viscous solutions can be explained by a simple diffusion model. This linear relationship is broken when a dimer conformation of the fluorophore starts to form, which in turn influences the emission intensity of the fluorophore.     

Fluorescence Origin of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) Bound to Proteins

6,P-toluidinylnaphthalene-2-sulfonate (TNS) is a highly fluorescent molecule when dissolved in a low polarity medium or when bound to proteins. The aim of the present work is to explain origin of this fluorescence, to find out how the medium (solvent, protein matrix) affects fluorescence observables such as lifetimes and spectra and finally to put into evidence possible relation that exists between these observables and fluorophore structure. To achieve our goal we performed studies on TNS dissolved in ethanol, at high concentrations in water (aggregated form) and bound to proteins. Our experiments allowed us to find out that TNS in the three environments has different structures. Presence of three lifetimes observed in proteins and in water instead of one lifetime found in ethanol can be assigned to the high contact between TNS molecules. Our results are discussed in terms of solvent polarity and interaction within fluorophore molecules bound to proteins.     

New Perspectives of Fluorescence Correlation Spectroscopy

The principle of fluorescence correlation spectroscopy is outlined. The technique has been applied to a mutant of the well-known green fluorescent protein. A comparative study has been made with time-resolved fluorescence anisotropy. The latter experiment shows that the fluorophore is rigidly bound inside the protein matrix follows the rotation of the whole protein and does not show any fast restricted motion. It is evident from fluorescence correlation spectroscopy that some excited-state reaction plays a role, since the autocorrelation traces show a significant effect on the incident laser power. Other potential applications of fluorescence correlation spectroscopy are presented as taken from very recent publications.     

异丙醇-水配合液的荧光偏振和时间分辨特性

研究了紫外光照射下异丙醇-水配合液的偏振荧光光谱,以及不同荧光峰处光子强度随时间的衰变过程,计算了偏振度并讨论了其偏振特性,测试了不同峰位对应的荧光寿命并分析了其荧光发射特性.结果表明,异丙醇-水配合液在紫外光激励下发射的荧光为具有确定分子取向的部分偏振光,偏振度和各向异性度分别为0.542和0.441.在波长为220...     

Fluorescence probes of viscosity: A comparative study of the fluorescence anisotropy decay of perylene and 3,9-dibromoperylene in glycerol

The authors compare the results of fluorescence anisotropy decay measurements for glycerol solutions of perylene with those of 3,9-dibromoperylene (DBP). For both molecules a good linear dependence is observed between the glycerol viscosity (varied by temperature) and the longer rotational correlation time obtained as a result of a global (using data obtained at 256- and 430-nm excitation wavelengths) biexponential analysis of the fluorescence anisotropy decay, at least in the range of 7–60 P for perylene and 4–60 P for DBP. This significantly extends the reported range of 0.5 to 150 cP investigated by Williams and Ben-Amotz [1] with the probe BTBP.     

Domain Formation in DODAB–Cholesterol Mixed Systems Monitored via Nile Red Anisotropy

The effect of the cholesterol (ch) on liposomes composed of the cationic lipid dioctadecyldimethylammonium bromide (DODAB) was assessed by studying both the steady-state and time-resolved fluorescence anisotropy of the dye Nile Red. The information obtained combined with analysis of the steady-state emission and fluorescence lifetime of Nile Red (NR) for different cholesterol concentrations (5–50%) elucidated the presence of “condensed complexes” and cholesterol-rich domains in these mixed systems. The steady-state fluorescence spectra were decomposed into the sum of two lognormal emissions, emanating from two different states, and the effect of temperature on the anisotropy decay of Nile Red for different cholesterol concentrations was observed. At room temperature, the time-resolved anisotropy decays are indicative of NR being relatively immobile (manifest by a high r _∞ value). At higher temperature, rotational times ca. 1 ns were obtained throughout and a trend in increasing hindrance was seen with increase of Ch content.